Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry.

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine-alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate-linked methylcyclopropene reporter that is well suited for the inverse-electron-demand Diels-Alder (DAinv ) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv , photoclick, and copper-free click chemistry.

Dienophile-Modified Mannosamine Derivatives for Metabolic Labeling of Sialic Acids: A Comparative Study.

Sialic acids play an important role in numerous cell adhesion processes, and sialylation levels are known to be altered under certain pathogenic conditions, such as cancer. Metabolic glycoengineering with mannosamine derivatives is a convenient way to introduce non-natural chemical reporter groups into sialylated glycoconjugates, offering the opportunity to label sialic acids by using bioorthogonal ligation chemistry. The labeling intensity depends not only on the rate of the ligation reaction but also on the extent to which the natural sialic acids are replaced by the modified ones; that is, the incorporation efficiency. Here, we present a comparative study of eight mannosamine derivatives featuring terminal alkenes as chemical reporter groups that can be labeled by an inverse-electron-demand Diels-Alder (DAinv) reaction. The derivatives differed in chain length as well as the type of linkage (carbamates, amides, and a urea) that connects the terminal alkene to the sugar. As a general trend, increasing chain lengths resulted in higher DAinv reactivity and, at the same time, reduced incorporation efficiency. Carbamates were better accepted than amides with the same chain length; nevertheless, the latter resulted in more intense cell-surface staining, visible by live-cell fluorescence microscopy. A urea derivative was also shown to be accepted.

Exploring the Potential of Norbornene-Modified Mannosamine Derivatives for Metabolic Glycoengineering.

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse-electron-demand Diels-Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene-modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

Visualization of Protein-Specific Glycosylation inside Living Cells.

Protein glycosylation is a ubiquitous post-translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein-specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels-Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan-anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).

Rapid labeling of metabolically engineered cell-surface glycoconjugates with a carbamate-linked cyclopropene reporter.

Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel N-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels-Alder reaction with inverse electron demand. We demonstrate that the cell's biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3-tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels-Alder reaction with inverse electron demand and a strain-promoted azide-alkyne cycloaddition which are carried out simultaneously in a single step.

Terminal alkenes as versatile chemical reporter groups for metabolic oligosaccharide engineering.

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.

Expanding the scope of cyclopropene reporters for the detection of metabolically engineered glycoproteins by Diels-Alder reactions.

Monitoring glycoconjugates has been tremendously facilitated by the development of metabolic oligosaccharide engineering. Recently, the inverse-electron-demand Diels-Alder reaction between methylcyclopropene tags and tetrazines has become a popular ligation reaction due to the small size and high reactivity of cyclopropene tags. Attaching the cyclopropene tag to mannosamine via a carbamate linkage has made the reaction even more efficient. Here, we expand the application of cyclopropene tags to N-acylgalactosamine and N-acylglucosamine derivatives enabling the visualization of mucin-type O-glycoproteins and O-GlcNAcylated proteins through Diels-Alder chemistry. Whereas the previously reported cyclopropene-labeled N-acylmannosamine derivative leads to significantly higher fluorescence staining of cell-surface glycoconjugates, the glucosamine derivative gave higher labeling efficiency with protein preparations containing also intracellular proteins.

Distinct CCR7 glycosylation pattern shapes receptor signaling and endocytosis to modulate chemotactic responses.

The homeostatic chemokines CCL19 and CCL21 and their common cognate chemokine receptor CCR7 orchestrate immune cell trafficking by eliciting distinct signaling pathways. Here, we demonstrate that human CCR7 is N-glycosylated on 2 specific residues in the N terminus and the third extracellular loop. Conceptually, CCR7 glycosylation adds steric hindrance to the receptor N terminus and extracellular loop 3, acting as a "swinging door" to regulate receptor sensitivity and cell migration. We found that freshly isolated human B cells, as well as expanded T cells, but not naïve T cells, express highly sialylated CCR7. Moreover, we identified that human dendritic cells imprint T cell migration toward CCR7 ligands by secreting enzymes that deglycosylate CCR7, thereby boosting CCR7 signaling on T cells, permitting enhanced T cell locomotion, while simultaneously decreasing receptor endocytosis. In addition, dendritic cells proteolytically convert immobilized CCL21 to a soluble form that is more potent in triggering chemotactic movement and does not desensitize the receptor. Furthermore, we demonstrate that soluble CCL21 functionally resembles neither the CCL19 nor the CCL21 phenotype but acts as a chemokine with unique features. Thus, we advance the concept of dendritic cell-dependent generation of micromilieus and lymph node conditioning by demonstrating a novel layer of CCR7 regulation through CCR7 sialylation. In summary, we demonstrate that leukocyte subsets express distinct patterns of CCR7 sialylation that contribute to receptor signaling and fine-tuning chemotactic responses.

Synthesis and X-ray structure analysis of a heptacoordinate titanium(IV)-bis-chelate with enhanced in vivo antitumor efficacy.

Chelate stabilization of a titanium(IV)-salan alkoxide by ligand exchange with 2,6-pyridinedicarboxylic acid (dipic) resulted in heptacoordinate complex 3 which is not redox-active, stable on silica gel and has increased aqueous stability. 3 is highly toxic in HeLa S3 and Hep G2 and has enhanced antitumor efficacy in a mouse cervical-cancer model.