Azido Sugars Research Articles

Process Development for the Enzymatic Gram-Scale Production of the Unnatural Nucleotide Sugar UDP-6-Azido-GalNAc.

Azido sugars hold great promise as substrates in numerous click-chemistry applications. However, the synthesis of activated azido sugars is limited by cost and complexity. Conventional chemical activation methods are intricate and time-consuming. In response, we have developed a process for the large-scale production of UDP-6-azido-GalNAc through enzymatic nucleotide sugar synthesis on a gram scale. Our optimization strategies encompassed refining the process parameters of an enzyme cascade featuring NahK from Bifidobacterium longum and AGX1 from Homo sapiens. Using the repetitive-batch-mode technology, we synthesized up to 2.1 g of UDP-6-azido-GalNAc, achieving yields up to 97 % in five consecutive batch cycles using a single enzyme batch. The synthesis process demonstrated to have total turnover numbers (TTNs) between 4.4-4.8 g of product per gram of enzyme (gP/gE) and STYs ranging from 1.7-2.4 g per liter per hour (g*L-1*h-1). By purification of a product solution pool containing 2.6 g (4.1 mmol) UDP-6-azido-GalNAc, 2.1 g (2,122.1 mg) UDP-6-azido-GalNAc (sodium salt) with a purity of 99.96 % (HPLC) were obtained. The overall recovery after purification was 81 % (3.32 mmol). Our work establishes a robust production platform for the gram-scale synthesis of unnatural nucleotide sugars, opening new avenues for applications in glycan engineering.

Programmed immobilization of living cells using independent click pairs

Therapeutic devices incorporating living cells or tissues have been intensively investigated for applications in tissue engineering and regenerative medicine. Because many biological processes are governed by spatially dependent signals, programmable immobilization of materials is crucial for manipulating multiple types of cells. In this study, click chemistry substrates were introduced onto the surfaces of cells and cover glass, and the cells were fixed on the cover glass via covalent bonds for selective cell deposition. Azide group (Az)-labeled living cells were prepared by metabolic labeling with azido sugars. Following the introduction of Az, TCO (trans-cyclooctene) was metabolically labeled into the living cells by reacting with TCO-DBCO (dibenzocyclooctyne). Az and TCO in the cells were detected using DBCO-FAM (fluorescein)and tetrazine-Cy3, respectively. The mixture of Az-labeled green fluorescent protein HeLa cells and TCO-labeled red fluorescent protein HeLa cells was reacted in a culture dish in which three different cover glasses, DBCO-, tetrazine-, or methyl-coated, were added. Az- or TCO-labeled cells could be immobilized in a functional group-dependent manner. Next, tetrazine-labeled cells were incubated on TCO- or Az-labeled cell layers instead of cover glass. Functional group-dependent immobilization was also achieved in the cell layer. Introducing substrates for the click reaction could achieve cell-selective immobilization on different patterned glass surfaces, as well as cell-cell immobilization.

Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis

BackgroundNutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2α O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear.MethodsWe used mass spectrometry to study the relationship between O-GlcNAcylation and Arg availability in breast cancer BT-549 cells. We validated eIF2α O-GlcNAcylation through site-specific mutagenesis and azido sugar N-azidoacetylglucosamine-tetraacylated labeling. We then evaluated the effect of eIF2α O-GlcNAcylation on cell recovery, migration, accumulation of reactive oxygen species (ROS), and metabolic labeling during protein synthesis under different Arg conditions.ResultsOur research identified eIF2α, eIF2β, and eIF2γ, as key O-GlcNAcylation targets in the absence of Arg. We found that O-GlcNAcylation of eIF2α plays a crucial role in regulating antioxidant defense by suppressing the translation of the enzyme HO-1 during Arg limitation. Our study showed that O-GlcNAcylation of eIF2α at specific sites suppresses HO-1 translation despite high levels of HMOX1 transcription. We also found that eliminating eIF2α O-GlcNAcylation through site-specific mutagenesis improves cell recovery, migration, and reduces ROS accumulation by restoring HO-1 translation. However, the level of the metabolic stress effector ATF4 is not affected by eIF2α O-GlcNAcylation under these conditions.ConclusionsOverall, this study provides new insights into how ArgS fine-tunes the control of translation initiation and antioxidant defense through eIF2α O-GlcNAcylation, which has potential biological and clinical implications.

Experimental and Theoretical Studies on Acid Corrosion Inhibition of API 5L X70 Steel with Novel 1-N-α-d-Glucopyranosyl-1H-1,2,3-Triazole Xanthines.

A series of novel 1-N-α-d-glucopyranosyl-1H-1,2,3-triazole xanthines was synthesized from azido sugars (glucose, galactose, and lactose) and propargyl xanthines (theophylline and theobromine) using a typical copper (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition. The corrosion inhibition activities of these new carbohydrate-xanthine compounds were evaluated by studying the corrosion of API 5 L X70 steel in a 1 M HCl medium. The results showed that, at 10 ppm, a 90% inhibition efficiency was reached by electrochemical impedance spectroscopy. The inhibitory efficiency of these molecules is explained by means of quantum chemical calculations of the protonated species with the solvent effect, which seems to better represent the actual situation of the experimental conditions. Some quantum chemical parameters were analyzed to characterize the inhibition performance of the tested molecules.

High-throughput screening of glycosynthases using azido sugars for oligosaccharides synthesis.

Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar. We created a diverse library of fucosynthase mutants using semi-random and random error prone mutagenesis and then identified improved fucosynthase mutants with desired activity using two distinct screening methods developed by our group to detect glycosynthase activity (i.e., by detecting azide formed upon completion of fucosynthase reaction); (a) pCyn-GFP regulon method, and (b) Click chemistry method. Finally, we provide some proof-of-concept results illustrating the utility of both these screening methods to rapidly detect products of glycosynthase reactions involving azido sugars as donor groups.

Azido Groups Hamper Glycan Acceptance by Carbohydrate Processing Enzymes.

Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structure of an azide compared to a hydroxyl. To quantitatively assess how well azides are accommodated, we have used glycosidases as tractable model enzyme systems reflecting what would also be expected for glycosyltransferases and other sugar binding/modifying proteins. In this vein, specificity constants have been measured for the hydrolysis of a series of azidodeoxy glucosides and N-acetylhexosaminides by a large number of glycosidases produced from expressed synthetic gene and metagenomic libraries. Azides at secondary carbons are not significantly accommodated, and thus, associated substrates are not processed, while those at primary carbons are productively recognized by only a small subset of the enzymes and often then only very poorly. Accordingly, in the absence of careful controls, results obtained with azide-modified sugars may not be representative of the situation with the natural sugar and should be interpreted with considerable caution. Azide incorporation can indeed provide a useful tool to monitor and detect glycosylation, but careful consideration should go into the selection of sites of azide substitution; such studies should not be used to quantitate glycosylation or to infer the absence of glycosylation activity.

Synthesis of 1,4‐Disubstituted 1,2,3‐Triazoles from Terminal Vinyl Sulfones in Ionic Liquid: A Metal‐Free Eliminative Azide‐Olefinic Cycloaddition Route to Triazolyl Carbohydrates and Triazole‐linked Bissaccharides

AbstractSeveral terminal vinyl sulfones, derived from styrene epoxide and monomesylated glycerol were reacted with benzylazide in a metal‐free condition to afford regioselectively 1,4‐disubstituted 1,2,3‐triazoles (1,4‐DTs) via Eliminative Azide‐Olefinic Cycloaddition (EAOC) reactions. The stereoelectronic repulsions between the aryl/alkyl groups attached to the azido and vinyl sulfone functionalities play a significant role in deciding the outcome of the reactions. The N, N‐dimethyl ethanolammonium formate‐water mixture consisting of a biodegradable ionic liquid turned out to be the best solvent system to reduce significantly the reaction time and increase the yield of products in almost all cases. Furthermore, a pair of exocyclic terminal vinyl sulfone‐modified carbohydrates were prepared from protected‐glucofuranoside and ‐allofuranoside. These carbohydrates were reacted under the EAOC conditions with a host of organic azides to afford the corresponding 1,4‐disubstituted 1,2,3‐triazole modified carbohydrates. Azido sugars were also reacted with terminal vinyl sulfone modified carbohydrates under similar conditions to afford 1,4‐disubstituted 1,2,3‐triazole linked bissacharides. This powerful strategy serves as a potential yet practical route for delivering two carbon unit (required for the C4‐C5 of the 1,2,3‐triazole ring) directly connected to sugar body in place of a sugar alkyne.

Efficient Synthesis of Azido Sugars Using Fluorosulfuryl Azide Diazotransfer Reagent**

AbstractAzide‐containing sugars are important tools for the synthesis of biologically relevant 1,2‐cis glycosides and for bioconjugation chemistry. Previous strategies to install a non‐participating C2‐azido functionality use harsh conditions and long reaction times. Herein, we report the synthesis of azido sugars using fluorosulfuryl azide (FSO2N3; 1) with a Cu(II) catalyst as a safe and efficient diazotransfer reagent. Common hexosamine substrates were converted to 2‐azido‐2‐deoxy sugars in less than 5 minutes in quantitative yield. Glycosyl donors with orthogonal protecting groups were readily prepared from these azido sugars with good overall yield and a single column purification. The diazotransfer protocol was also efficiently applied to other amino sugar derivatives, including aminoglycosides, substrates with amine‐containing linkers, and it is orthogonal to O‐sulfation. This optimized method will expand access to important non‐participating C2‐azido protecting groups and other azido sugar derivatives.

Chemical and chemoenzymatic routes to bridged homoarabinofuranosylpyrimidines: Bicyclic AZT analogues.

Conformationally restricted diastereomeric homoarabinofuranosylpyrimidines (AZT analogue), i.e., (5′R)-3′-azido-3′-deoxy-2′-O,5′-C-bridged-β-ᴅ-homoarabinofuranosylthymine and -uracil had been synthesized starting from diacetone ᴅ-glucofuranose following chemoenzymatic and chemical routes in 34–35% and 24–25% overall yields, respectively. The quantitative and diastereoselective acetylation of primary hydroxy over two secondary hydroxy groups present in the key nucleoside precursor was mediated with Lipozyme® TL IM in 2-methyltetrahydrofuran following a chemoenzymatic pathway. Whereas, the protection of the primary hydroxy over the lone secondary hydroxy group in the key azido sugar precursor was achieved using bulky tert-butyldiphenylsilyl chloride (TBDPS-Cl) in pyridine in 92% yield following a chemical synthetic pathway. The chemoenzymatic method was found to be superior over the chemical method in respect of the number of synthetic steps and overall yield of the final product.

Design and synthesis of eugenol/isoeugenol glycoconjugates and other analogues as antifungal agents against Aspergillus fumigatus.

Glycoconjugates are biologically significant molecules as they tend to serve a wide range of intra- and extra-cellular processes depending on their size and complexity. The secondary metabolites of the plant Myristica fragrans, eugenol and isoeugenol, have shown antifungal activities (IC50 1900 μM). Therefore, we envisioned that glycoconjugates based on these two scaffolds could prove to be potent antifungal agents. Triazole-containing compounds have shown prominent activities as antifungal agents. Based on this, we opined that a Cu(i) catalyzed click reaction could serve as the bridging tool between a eugenol/isoeugenol moiety and sugars to synthesize eugenol/isoeugenol based glycoconjugates. In our present work, we have coupled propargylated eugenol/isoeugenol and azido sugar to furnish eugenol/isoeugenol based glycoconjugates. In another approach, we have carried out hydroxylation of the double bond of eugenol and subsequent azidation of a primary alcohol followed by intramolecular coupling reactions leading to various other analogues. All the synthesized compounds were assayed against an opportunistic pathogenic fungus, Aspergillus fumigatus. Among the synthesized compounds, two analogues have exhibited significant antifungal activities with IC50 values of 5.42 and 9.39 μM, respectively. The study suggested that these two analogues inhibit cell wall-associated melanin hydrophobicity along with the number of conidia. The synthesized compounds were found to be non-cytotoxic to an untransformed cell line.

Metabolic Labeling of Primary Neurons Using Carbohydrate Click Chemistry.

Glycans play an important role in many neuronal processes, such as neurotransmitter release and reuptake, cell-cell communication and adhesion, modulation of ion channel activity, and immune function. Carbohydrate click chemistry is a powerful technique for studying glycan function and dynamics in vitro, in vivo, and ex vivo. Here, we use commercially available synthetic tetraacetylated azido sugars, copper and copper-free click chemistry to metabolically label and analyze primary rat cortical neurons. In addition, we use high resolution confocal and STED microscopy to image and analyze different forms of glycosylation in ultrahigh resolution. We observe different patterns of GlcNAz, GalNAz, and ManNAz distribution at different stages of neuronal development. We also observe highly sialylated structureson the neuronal plasma membrane, which warrant further investigation.

Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity.

Engineering of carbohydrate-active enzymes such as glycosynthases to enable chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable ultrahigh-throughput screening methods capable of robustly detecting either starting substrates or end-products of the glycosidic bond formation reaction. Currently, there are limited screening methods available for rapid and highly sensitive single-cell-based screening of glycosynthase enzymes employing azido sugars as activated donor glycosyl substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides as substrates versus free inorganic azides as reaction products that facilitated an ultrahigh-throughput in vivo single-cell-based assay of glycosynthase activity. This assay was developed based on the distinct differences observed in relative fluorescence intensity of the triazole-containing fluorophore product formed during the click-chemistry reaction of organic glycosyl azides versus inorganic azides. This discovery formed the basis for proof of concept validation of a directed evolution methodology for screening and sorting glycosynthase mutants capable of synthesis of targeted fucosylated oligosaccharides. Our screening approach facilitated fluorescence-activated cell sorting of an error-prone polymerase chain reaction-based mutant library of fucosynthases expressed in Escherichia coli to identify several novel mutants that showed increased activity for β-fucosyl azide-activated donor sugars toward desired acceptor sugars (e.g., pNP-xylose and lactose). Finally, we discuss avenues for improving this proof of concept in vivo assay method to identify better glycosynthase mutants and further demonstrate the broader applicability of this screening methodology for synthesis of bespoke glycans.

Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor.

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering.

Detection of azide-tagged biomolecules (e.g., azido sugars) inside living cells using "click" chemistry has been revolutionary to the field of chemical biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called cyn regulon, and complementary Escherichia coli engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes. The engineered cyn azide operon allowed highly tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01-5 mM) and rapidly induced GFP expression by over 600-fold compared to the uninduced control. Next, we showcase the superior performance of this engineered cyn-operon over the classical lac-operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitating in vivo activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.

Sugar-coating dendritic cells.

In situ labeling with azido sugars enables in vivo immunomodulation of dendritic cells.

Repurposing Intracellular Fluorescent Biosensors to Visualize Extracellular Fluxes

With dozens of fluorescent biosensors designed to detect intracellular ions and metabolites, we sought an approach to repurpose these biosensors to visualize extracellular fluxes. Because most fluorescent ion and metabolite biosensors are optimized in bacteria and purified with nickel NTA beads, we synthesized a water soluble Ni2+-tris-NTA reagent that covalently modifies a cell's glycocalyx metabolically-labeled with an unnatural azido sugar. Subsequent cell surface labeling with a purified polyhistidine-tagged biosensor enabled visualization of ion channel and membrane transporter activity at the plasma membrane. The synthesis of the reagent, the labeling procedure, and fluorescent visualization of extracellular fluxes using the repurposed intracellular biosensors will be presented.

Robustness in glycosylation systems: effect of modified monosaccharides, acceptor decoys and azido sugars on cellular nucleotide-sugar levels and pattern of N-linked glycosylation.

Small molecule monosaccharide analogs (e.g. 4F-GlcNAc, 4F-GalNAc) and acceptor decoys (e.g. ONAP, SNAP) are commonly used as metabolic glycoengineering tools to perturb molecular and cellular recognition processes. Azido-derivatized sugars (e.g. ManNAz, GlcNAz, GalNAz) are also used as bioorthogonal probes to assay the glycosylation status of cells and tissue. With the goal of obtaining a systems-level understanding of how these compounds work, we cultured cells with these molecules and systematically evaluated their impact on: (i) cellular nucleotide-sugar levels, and (ii) N-linked glycosylation. To this end, we developed a streamlined, simple workflow to quantify nucleotide-sugar levels using amide-based hydrophilic interaction liquid chromatography (HILIC) separation followed by negative-mode electrospray ionization mass spectrometry (ESI-MS/MS) using an Orbitrap detector. N-Glycans released from cells were also procainamide functionalized and quantified using positive-mode ESI-MS/MS. Results show that all tested compounds changed the baseline nucleotide-sugar levels, with the effect being most pronounced for the fluoro-HexNAc compounds. These molecules depressed UDP-HexNAc levels in cells by up to 80%, while concomitantly elevating UDP-4F-GalNAc and UDP-4F-GlcNAc. While the measured changes in nucleotide-sugar concentration were substantial in many cases, their impact on N-linked glycosylation was relatively small. This may be due to the high nucleotide-sugar concentrations in the Golgi, which far exceed the KM values of the glycosylating enzymes. Thus, the glycosylation system output exhibits 'robustness' even in the face of significant changes in cellular nucleotide-sugar concentrations.

The distinct difference in azido sugar metabolic rate between neural stem cells and fibroblasts and its application for decontamination of chemically induced neural stem cells.

In our report, we found a distinct difference in azido sugar metabolic rate between neural stem cells and fibroblasts, which can be used for selective removal of fibroblasts from neural stem cell mixtures. Chemically induced neural stem cells (ciNSCs) serve as a highly valuable source of NSCs. Incompletely induced fibroblasts could interfere with ciNSC differentiation and become tumorigenic. Herein, we applied our method for the decontamination of ciNSCs and it exhibited excellent selectivity for ciNSCs. The results demonstrate that the ciNSC population can be efficiently purified to 98.1%. As far as we know, this is the highest purity obtained so far. We envision that, in the future, our method could be used as a safe, effective, and chemically-defined tool for decontaminating ciNSCs in both fundamental research and clinical stem cell therapy.

Fluorometric visualization of mucin 1 glycans on cell surfaces based on rolling-mediated cascade amplification and CdTe quantum dots.

A rolling-mediated cascade (RMC) amplification strategy is described for improved visualization of profiling glycans of mucin 1 (MUC 1) on cell surfaces. CdTe quantum dots (QDs) are used as fluorescent labels. The RMC based amplification allows even distinct glycoforms of MUC1 to be visualized on the surface of MCF-7 cell via an amplified Förster resonance energy transfer (FRET) imaging strategy that works at excitation/emission wavelengths of 345/610nm. This is achieved by utilizing antibody against MUC1 modified with the fluorescent label 7-amino-4-methylcoumarin-3-acetic acid (AMCA) as the energy donor in FRET. The QDs (used to label surface glycans) act as acceptors. N-Azidoacetylgalactosamine-Acetylated (Ac4GalNAz) as a non-natural azido sugar, can be incorporated into the glycans of the cell surface, which can promote further labeling. The method has the advantage of only requiring a small amount of non-natural sugar to be introduced in metabolic glycan labeling since too much of an artificial sugar will interfere with the physiological functions of cells. Graphical abstract Schematic for theDNA rolling-mediated cascade (RMC)-assisted metabolic labeling of cell surface glycans by using CdTe quantum dots as labels and an intramolecular amplified FRET strategy for imaging glycans on a specific glycosylated protein, MUC1.

VISUALIZATION AND CHARACTERIZATION OF EXOSOMES IN BREAST CANCER CELLS

Objectives Exosomes are extracellular vesicles of endocytic origin with a size range of 40-150 nm and a lipid bilayer membrane. Though exosomes are known as dynamic mediators of intercellular communication, its characteristics and function have not been fully studied. In this report, we used a metabolic labeling method to prepare fluorescent exosomes to investigate the characteristics and the movement of exosomes derived from various breast cancer cells. Findings MCF-7 and MDA-MB-231 cells were treated with three types of azido sugars, Ac4ManNAz, Ac4GalNAz, or Ac4GlcNAz (50 mM) for 3 days to produce the azide (-N3) containing exosomes through metabolic glycosylation. It is confirmed that the azido sugar decorated exosomes were able to maintain their original characteristics such as size, lipid bilayer morphology, and protein profile. The exosomes prepared with Ac4ManNAz have shown the highest labeling efficiency with ADIBO-Cy3 fluorescence dye in MCF-7, MDA-MB-231, BT-549 and MDA-MB-468 cells at 17.2%, 14.8%, 13.2% and 14.9% respectively. To study their uptake capability, the labeled exosomes from different origins were incubated with a panel of breast cancer cells, including MCF-7, MDA-MB-231, MDA-MB-468 and BT-549, and normal NIH/3T3 fibroblast cells for 24 hours. The cells were then evaluated by fluorescence microscopic imaging and flow cytometry. It was observed that exosomes from different origins have a different uptake efficiency, suggesting that each exosome may have its unique navigation systems. Furthermore, the cells which have aggressive metastatic potential, such as MDA-MB-231 showed a better pickup of all exosomes. In contrast, the exosomes released by MDA- MB-468 showed higher loading in five kinds of cells. Conclusions Our development has demonstrated an effective labeling method that can facilitate exosome research by providing a new way of quantification and tracking in vitro and potentially in vivo studies.