The use of sugars with bioorthogonal functionalities introduced into living cells or added enzymatically has proven to be a valuable research tool for defining the functional roles of glycosylation in cellular systems. An alternative chemical approach to introducing bioorthogonal groups to specific sugars is described herein, one that specifically targets biologically important terminal sialic acid isomers attached to glycoconjugates in tissues and cells on slides. The most common sialic acid isomers are attached in either α2,3, α2,6, or α2,8 linkages to glycan termini which confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences using standard mass spectrometry approaches. Herein, a sequential double amidation chemical derivatization strategy is described that results in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3 and α2,8-linked sialic acids in the second amidation step. This allows for targeted detection of cells in tissue or individually on glass slides using mass spectrometry imaging or microscopy approaches, as well as distinct mass shifts from α2,6-linked amidated isomers created in the first amidation reaction step. Use of an alkyne-amine like propargylamine as the second amidation reagent introduces different mass shifts for α2,3-linked and α2,8-linked sialic acids. Use of an azide-amine with a poly-ethylene glycol linker introduces a sterically approachable azide group on α2,3-lined and α2,8-linked sialic acids. This azide group can be targeted for click chemistry reactions with biotin-alkyne for streptavidin-conjugated imaging by histochemical stains and fluorescence. Detailed protocols for several example uses are provided.

N-glycosylation is an abundant post-translational modification of most cell-surface proteins. N-glycans play a crucial role in cellular functions like protein folding, protein localization, cell–cell signaling, and immune detection. As different tissue types display different N-glycan profiles, changes in N-glycan compositions occur in tissue-specific ways with development of disease, like cancer. However, no comparative atlas resource exists for documenting N-glycome alterations across various human tissue types, particularly comparing normal and cancerous tissues. In order to study a broad range of human tissue N-glycomes, N-glycan targeted MALDI imaging mass spectrometry was applied to custom formalin-fixed paraffin-embedded tissue microarrays. These encompassed fifteen human tissue types including bladder, breast, cervix, colon, esophagus, gastric, kidney, liver, lung, pancreas, prostate, sarcoma, skin, thyroid, and uterus. Each array contained both normal and tumor cores from the same pathology block, selected by a pathologist, allowing more in-depth comparisons of the N-glycome differences between tumor and normal and across tissue types. Using established MALDI-IMS workflows and existing N-glycan databases, the N-glycans present in each tissue core were spatially profiled and peak intensity data compiled for comparative analyses. Further structural information was determined for core fucosylation using endoglycosidase F3, and differentiation of sialic acid linkages through stabilization chemistry. Glycan structural differences across the tissue types were compared for oligomannose levels, branching complexity, presence of bisecting N-acetylglucosamine, fucosylation, and sialylation. Collectively, our research identified the N-glycans that were significantly increased and/or decreased in relative abundance in cancer for each tissue type. This study offers valuable information on a wide scale for both normal and cancerous tissues, serving as a reference for future studies and potential diagnostic applications of MALDI-IMS.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

N-linked glycosylation plays an important role in both the innate and adaptive immune response through the modulation of cell surface receptors as well as general cell-to-cell interactions. The study of immune cell N-glycosylation is gaining interest but is hindered by the complexity of cell-type-specific N-glycan analysis. Analytical techniques such as chromatography, LC-MS/MS, and the use of lectins are all currently used to analyze cellular glycosylation. Issues with these analytical techniques include poor throughput, which is often limited to a single sample at a time, lack of structural information, the need for a large amount of starting materials, and the requirement for cell purification, thereby reducing their feasibility for N-glycan study. Here, we report the development of a rapid antibody array-based approach for the capture of specific nonadherent immune cells coupled with MALDI-IMS to analyze cellular N-glycosylation. This workflow is adaptable to multiple N-glycan imaging approaches such as the removal or stabilization and derivatization of terminal sialic acid residues providing unique avenues of analysis that have otherwise not been explored in immune cell populations. The reproducibility, sensitivity, and versatility of this assay provide an invaluable tool for researchers and clinical applications, significantly expanding the field of glycoimmunology.

Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.

Cholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP) related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide. IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.

Oligosaccharides equipped with amine-containing linkers can be conjugated to carrier proteins using squaric acid chemistry. In a two-step process, a squarate derivative of such oligosaccharide is formed first, which is followed by its reaction with a protein carrier. Monitoring of the conjugation reaction is achieved by SELDI-TOF-MS or MALDI-TOF-MS. This experimentally simple procedure yields desired glycoconjugates in high yields and with reproducible hapten-protein ratios.

The sequence α‐Col p ‐(1→2)‐4,6‐ P ‐β‐ d ‐Gal p ‐(1→3)‐[α‐Col p ‐(1→4)]‐β‐ d ‐Glc p NAc‐(1→4)‐α‐ d ‐Gal p A‐(1→3)‐β‐ d ‐Qui p NAc constitutes the complete O‐specific polysaccharide (O‐antigen, O‐SP) of Vibrio cholerae O139. It was chemically synthesized in a linker‐equipped, conjugation‐ready form ( 7 ) and conjugated to a model protein carrier, bovine serum albumin. The preparation involved the synthesis of a tetrasaccharide intermediate sequence β‐ d ‐Gal p ‐(1→3)‐β‐ d ‐Glc p NAc‐(1→4)‐α‐ d ‐Gal p A‐(1→3)‐β‐ d ‐Qui p NAc→linker by coupling of two disaccharide intermediates followed by a stepwise, two single‐site colitosylations. The present synthesis is an alternative to the academic, small‐scale synthesis developed earlier where the final hexasaccharide assembly was achieved by simultaneous, two‐sites colitosylation. The approach reported herein comprises a few more synthetic steps but is experimentally less demanding, minimizes separation difficulties and is more convenient when tens of mgs of the final product is required. Syntheses of non‐phosphorylated and methyl ester analogues of 7 are also described.

Three pentasaccharides, two tetrasaccharides, and a trisaccharide fragment of the O-specific antigen of Vibrio cholerae O139 were synthesized by applying 1 + 1, 2 + 1, 3 + 1, and 4 + 1 coupling strategies. The most challenging tasks involved were the synthesis of the 1,2-cis-glycosidic linkage between galactose and the linker (spacer) molecule and final purification of the target multicharged substances. Difficulties with final deprotection by hydrogenation/hydrogenolysis caused by the presence of galacturonic acid were overcome by protecting the acid with a group inert to the treatment with hydrogen. Some intermediates described previously as incompletely characterized amorphous materials were obtained in the crystalline condition and were fully characterized for the first time.

Hyaluronan (HA) is an integral component of the extracellular matrix. Its interactions with a cell surface receptor CD44 has been shown to play important roles in a variety of biological events including cell proliferation and metastasis. As multivalent CD44-HA binding is critical for downstream signaling, compounds that can selectively disrupt the complex formation of HA polysaccharide with CD44 can serve as useful probes of CD44 mediated cellular events as well as potential leads for novel therapeutics. Herein, we report the synthesis of several series of HA conjugates to target the HA binding pocket of CD44. As a small library of HA disaccharide derivatives failed to exhibit any inhibitory activities, we focused on HA tetrasaccharide based analogs. Traditional synthetic strategies towards HA oligosaccharides involve the construction of backbone from the corresponding monosaccharide building blocks, which can be quite tedious. In order to expedite the synthesis, we designed a new synthetic route taking advantage of the ability of hyaluronidase to generate large quantities of HA tetrasaccharide through digestion of HA polysaccharides. The HA tetrasaccharide obtained was utilized to prepare multiple S-linked HA analogs bearing aromatic groups at the reducing end glycan. One such compound containing an m-benzyl phenyl moiety exhibited significant inhibition of CD44-HA binding. Our approach provides a new direction towards the design of HA based CD44 antagonists.

Glyconanoparticles are the subject of numerous literatures and are emerging for applications in biomedicine, glycoscience, and material science due to their unique properties. They are hybrid materials each of which contains a nanoparticle core surrounded by a carbohydrate shell. The core can be metallic, magnetic, and/or photoluminescent, while the shell may be composed of mono-, di-, oligo- and/or poly-saccharides. Sugars are attached to the surface of the nanoparticles through covalent or non-covalent interaction. Here, we give a brief summary of the synthetic methodologies adopted in the preparation of different types of glyconanoparticles, which can be utilized in various applications including catalysis, drug delivery, vaccine, imaging and study of carbohydrate-lectin interactions.

The preparation of 4',6'-O-benzylidenecellobiose has been improved and its p-methoxybenzylidene counterpart was prepared in a similar way. As with other alkylidene derivatives of sugars, the corresponding per-O-benzoates are versatile intermediates in carbohydrate synthesis, allowing, after hydrolytic cleavage or opening of the phenylidene ring, regioselective manipulation of hydroxyl groups in cellobiose, or access to various derivatives of lactose.

The preparation of 4′,6′- O -benzylidenecellobiose has been improved and its p -methoxybenzylidene counterpart was prepared in a similar way. As with other alkylidene derivatives of sugars, the corresponding per- O -benzoates are versatile intermediates in carbohydrate synthesis, allowing, after hydrolytic cleavage or opening of the phenylidene ring, regioselective manipulation of hydroxyl groups in cellobiose, or access to various derivatives of lactose.

Imaging and targeted delivery to macrophages are promising new approaches to study and treat a variety of inflammatory diseases such as atherosclerosis. In this manuscript, we have designed and synthesized iron oxide based magnetic nanoparticles bearing hyaluronic acid (HA) on the surface to target activated macrophages. The HA-coated nanoparticles were prepared through a co-precipitation procedure followed by postsynthetic functionalization with HA and fluorescein. The nanoparticles were characterized by transmission electron microscopy, thermogravimetric analysis, elemental analysis, dynamic light scattering, and high-resolution magic angle spinning NMR and were biocompatible with cells and colloidally stable in the presence of serum. The HA immobilized on the nanoparticles retained their specific biological recognition with the HA receptor CD44, which is present on activated macrophages in high-affinity forms. Cell uptake studies demonstrated significant uptake of HA nanoparticles by activated macrophage cell line THP-1, which enabled magnetic resonance imaging of THP-1 cells. The uptake of nanoparticles was found to be both HA and CD44 dependent. Interestingly, Prussian blue staining showed that the magnetite cores of the HA-coated nanoparticles were only transiently present inside the cells, thus reducing the potential concerns of nanotoxicity. Furthermore, fluorescein on the nanoparticle was found to be delivered to the cell nucleus. Therefore, with further development, these HA functionalized magnetic nanoparticles can potentially become a useful carrier system for molecular imaging and targeted drug delivery to activated macrophages.

The chemical synthesis of a hyaluronic acid decasaccharide using the preactivation-based chemoselective glycosylation strategy is described. Assembly of large oligosaccharides is generally challenging due to the increased difficulties in both glycosylation and deprotection. Indeed, the same building blocks previously employed for hyaluronic acid hexasaccharide syntheses failed to yield the desired decasaccharide. After extensive experimentation, the decasaccharide backbone was successfully constructed with an overall yield of 37% from disaccharide building blocks. The trichloroacetyl group was used as the nitrogen protective group for the glucosamine units, and the addition of TMSOTf was found to be crucial to suppress the formation of trichloromethyl oxazoline side product and enable high glycosylation yield. For deprotections, the combination of a mild basic condition and the monitoring methodology using 1H NMR allowed the removal of all base-labile protective groups, which facilitated the generation of the fully deprotected HA decasaccharide.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.