Parametric Analysis of Donor Activation for Glycosylation Reactions.

The chemical synthesis of complex oligosaccharides relies on efficient and highly reproducible glycosylation reactions. The outcome of a glycosylation is contingent upon several environmental factors, such as temperature, acidity, the presence of residual moisture, as well as the steric, electronic, and conformational aspects of the reactants. Each glycosylation proceeds rapidly and with a high yield within a rather narrow temperature range. For better control over glycosylations and to ensure fast and reliable reactions, a systematic analysis of 18 glycosyl donors revealed the effect of reagent concentration, water content, protecting groups, and structure of the glycosyl donors on the activation temperature. With these insights, we parametrize the first step of the glycosylation reaction to be executed reliably and efficiently.

Synthesis of a heparan sulfate tetrasaccharide using automated glycan assembly.

Herein we utilise automated glycan assembly to complete solid-phase synthesis of defined heparan sulfate oligosaccharides, employing challenging D-glucuronate disaccharide donors. Using an orthogonally protected D-GlcN-α-D-GlcA donor, milligram-scale synthesis of a heparan sulfate tetrasaccharide is completed in 18% yield over five steps. Furthermore, orthogonal protecting groups enabled regiospecific on-resin 6-O-sulfation. This methodology provides an important benchmark for the rapid assembly of biologically relevant heparan sulfate sequences.

Phosphates as Assisting Groups in Glycan Synthesis.

In nature, phosphates are added to and cleaved from molecules to direct biological pathways. The concept was adapted to overcome limitations in the chemical synthesis of complex oligosaccharides. Phosphates were chemically placed on synthetic glycans to ensure site-specific enzymatic elongation by sialylation. In addition, the deliberate placement of phosphates helped to solubilize and isolate aggregating glycans. Upon traceless removal of the phosphates by enzymatic treatment with alkaline phosphatase, the native glycan structure was revealed, and the assembly of glycan nanostructures was triggered.

Heparan Sulfate-Mimicking Glycopolymers Bind SARS-CoV-2 Spike Protein in a Length- and Sulfation Pattern-Dependent Manner.

Heparan sulfate-mimicking glycopolymers, composed of glucosamine (GlcN)-glucuronic acid (GlcA) repeating units, bind to the receptor-binding subunit (S1) and spike glycoprotein (S) domains of the SARS-CoV-2 spike protein in a length- and sulfation pattern-dependent fashion. A glycopolymer composed of 12 repeating GlcNS6S-GlcA units exhibits a much higher affinity to the S1 protein (IC50 = 13 ± 1.1 nM) compared with the receptor-binding domain (RBD). This glycopolymer does not interfere in angiotensin-converting enzyme 2 binding of the RBD. Although this compound binds strongly to the S1/membrane-fusion subunit (S2) junction (KD = 29.7 ± 4.18 nM), it does not shield the S1/S2 site from cleavage by furin-a behavior contrary to natural heparin. This glycopolymer lacks iduronic acid, which accounts for 70% of heparin. Further, this compound, unlike natural heparin, is well defined in both sulfation pattern and length, which results in fewer off-target interactions with heparin-binding proteins. The results highlight the potential of using polymeric heparan sulfate (HS) mimetics for the therapeutic agent development.

Enabling Technologies in Carbohydrate Chemistry: Automated Glycan Assembly, Flow Chemistry and Data Science.

The synthesis of defined oligosaccharides is a complex task. Several enabling technologies have been introduced in the last two decades to facilitate synthetic access to these valuable biomolecules. In this concept, we describe the technological solutions that have advanced glycochemistry using automated glycan assembly, flow chemistry and data science as examples. We highlight how the synergies between these different technologies can further advance the field, with progress toward the realization of a self-driving lab for glycan synthesis.

VaporSPOT: Parallel Synthesis of Oligosaccharides on Membranes.

Automated chemical synthesis has revolutionized synthetic access to biopolymers in terms of simplicity and speed. While automated oligosaccharide synthesis has become faster and more versatile, the parallel synthesis of oligosaccharides is not yet possible. Here, a chemical vapor glycosylation strategy (VaporSPOT) is described that enables the simultaneous synthesis of oligosaccharides on a cellulose membrane solid support. Different linkers allow for flexible and straightforward cleavage, purification, and characterization of the target oligosaccharides. This method is the basis for the development of parallel automated glycan synthesis platforms.

Heparan Sulfate Mimicking Glycopolymer Prevents Pancreatic ß Cell Destruction and Suppresses Inflammatory Cytokine Expression in Islets under the Challenge of Upregulated Heparanase.

Diabetes is a chronic disease in which the levels of blood glucose are too high because the body does not effectively produce insulin to meet its needs or is resistant to insulin. ß Cells in human pancreatic islets produce insulin, which signals glucogen production by the liver and causes muscles and fat to uptake glucose. Progressive loss of insulin-producing ß cells is the main cause of both type 1 and type 2 diabetes. Heparan sulfate (HS) is a ubiquitous polysaccharide found at the cell surface and in the extracellular matrix (ECM) of a variety of tissues. HS binds to and assembles proteins in ECM, thus playing important roles in the integrity of ECM (particularly basement membrane), barrier function, and ECM-cell interactions. Islet HS is highly expressed by the pancreatic ß cells and critical for the survival of ß cells. Heparanase is an endoglycosidase and cleaves islet HS in the pancreas, resulting in ß-cell death and oxidative stress. Heparanase could also accelerate ß-cell death by promoting cytokine release from ECM and secretion by activated inflammatory and endothelial cells. We demonstrate that HS-mimicking glycopolymer, a potent heparanase inhibitor, improves the survival of cultured mouse pancreatic ß cells and protects HS contents under the challenge of heparanase in human pancreatic islets. Moreover, this HS-mimicking glycopolymer reduces the expression levels of cytokines (IL8, IL1ß, and TNFα) and the gene encoding Toll-like Receptor 2 (TLR2) in human pancreatic islets.

On resin synthesis of sulfated oligosaccharides.

Sulfated glycans are involved in many biological processes, making well-defined sulfated oligosaccharides highly sought molecular probes. These compounds are a considerable synthetic challenge, with each oligosaccharide target requiring specific synthetic protocols and extensive purifications steps. Here, we describe a general on resin approach that simplifies the synthesis of sulfated glycans. The oligosaccharide backbone, obtained by Automated Glycan Assembly (AGA), is subjected to regioselective sulfation and hydrolysis of protecting groups. The protocol is compatible with several monosaccharides and allows for multi-sulfation of linear and branched glycans. Seven diverse, biologically relevant sulfated glycans were prepared in good to excellent overall yield.

Towards a Systematic Understanding of the Influence of Temperature on Glycosylation Reactions.

Glycosidic bond formation is a continual challenge for practitioners. Aiming to enhance the reproducibility and efficiency of oligosaccharide synthesis, we studied the relationship between glycosyl donor activation and reaction temperature. A novel semi-automated assay revealed diverse responses of members of a panel of thioglycosides to activation at various temperatures. The patterns of protecting groups and the thiol aglycon combine to cause remarkable differences in temperature sensitivity among glycosyl donor building blocks. We introduce the concept of donor activation temperature to capture experimental insights, reasoning that glycosylations performed below this reference temperature evade deleterious side reactions. Activation temperatures enable a simplified temperature treatment and facilitate optimization of glycosyl donor usage. Isothermal glycosylation below the activation temperature halved the equivalents of building block required in comparison to the standard "ramp" regime used in solution- and solid-phase oligosaccharide synthesis to-date.

Expedited synthesis of mannose-6-phosphate containing oligosaccharides.

Currently, the reaction toolbox for the functionalization of glycans assembled on solid-phase is quite limited. Automated (1 h) and manual (overnight) phosphorylation protocols that enable the solid-phase synthesis of oligosaccharides containing up to two mannose-6-phosphates are presented. Automated glycan assembly expedited access to substrates and facilitated the screening of experimental conditions.

Microwave-Assisted Automated Glycan Assembly.

Automated synthesis of DNA, RNA, and peptides provides quickly and reliably important tools for biomedical research. Automated glycan assembly (AGA) is significantly more challenging, as highly branched carbohydrates require strict regio- and stereocontrol during synthesis. A new AGA synthesizer enables rapid temperature adjustment from -40 to +100 °C to control glycosylations at low temperature and accelerates capping, protecting group removal, and glycan modifications using elevated temperatures. Thereby, the temporary protecting group portfolio is extended from two to four orthogonal groups that give rise to oligosaccharides with up to four branches. In addition, sulfated glycans and unprotected glycans can be prepared. The new design reduces the typical coupling cycles from 100 to 60 min while expanding the range of accessible glycans. The instrument drastically shortens and generalizes the synthesis of carbohydrates for use in biomedical and material science.

Visible-Light-Mediated Oxidative Debenzylation Enables the Use of Benzyl Ethers as Temporary Protecting Groups.

The cleavage of benzyl ethers by catalytic hydrogenolysis or Birch reduction suffers from poor functional group compatibility and limits their use as a protecting group. The visible-light-mediated debenzylation disclosed here renders benzyl ethers temporary protective groups, enabling new orthogonal protection strategies. Using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a stoichiometric or catalytic photooxidant, benzyl ethers can be cleaved in the presence of azides, alkenes, and alkynes. The reaction time can be reduced from hours to minutes in continuous flow.

Automated Glycan Assembly in a Variable-Bed Flow Reactor Provides Insights into Oligosaccharide-Resin Interactions.

A pressure-based variable-bed flow reactor built for peptide synthesis and capable of real-time monitoring of resin swelling was adapted for automated glycan assembly. In the context of the solid-phase synthesis of several oligosaccharides, the coupling efficiencies, resin growth patterns, and saccharide solvation during the synthesis were determined. The presented work provides the first estimation of on-resin oligosaccharide solvation and an alternative technique to UV-vis monitoring.

Are Brønsted Acids the True Promoter of Metal-Triflate-Catalyzed Glycosylations? A Mechanistic Probe into 1,2-cis-Aminoglycoside Formation by Nickel Triflate.

Metal triflates have been utilized to catalytically facilitate numerous glycosylation reactions under mild conditions. In some methods, the metal triflate system provides stereocontrol during the glycosylation, rather than the nature of protecting groups on the substrate. Despite these advances, the true activating nature of metal triflates remains unclear. Our findings indicated that the in situ generation of trace amounts of triflic acid from metal triflates can be the active catalyst species in the glycosylation. This fact has been mentioned previously in metal triflate-catalyzed glycosylation reactions; however, a thorough study on the subject and its implications on stereoselectivity has yet to be performed. Experimental evidence from control reactions and 19F NMR spectroscopy have been obtained to confirm and quantify the triflic acid released from nickel triflate, for which it is of paramount importance in achieving a stereoselective 1,2-cis-2-amino glycosidic bond formation via a transient anomeric triflate. A putative intermediate resembling that of a glycosyl triflate has been detected using variable temperature NMR (1H and 13C) experiments. These observations, together with density functional theory calculations and a kinetic study, corroborate a mechanism involving triflic acid-catalyzed stereoselective glycosylation with N-substituted trifluoromethylbenzylideneamino protected electrophiles. Specifically, triflic acid facilitates formation of a glycosyl triflate intermediate which then undergoes isomerization from the stable α-anomer to the more reactive ß-anomer. Subsequent SN2-like displacement of the reactive anomer by a nucleophile is highly favorable for the production of 1,2-cis-2-aminoglycosides. Although there is a previously reported work regarding glycosyl triflates, none of these reports have been confirmed to come from the counter ion of the metal center. Our work provides supporting evidence for the induction of a glycosyl triflate through the role of triflic acid in metal triflate-catalyzed glycosylation reactions.

Real-time monitoring of solid-phase peptide synthesis using a variable bed flow reactor.

On-resin aggregation and incomplete amide bond formation are major challenges for solid-phase peptide synthesis that are difficult to be monitored in real-time. Incorporation of a pressure-based variable bed flow reactor into an automated solid-phase peptide synthesizer permitted real-time monitoring of resin swelling to determine amino acid coupling efficiency and on-resin aggregation.

Facile triflic acid-catalyzed α-1,2-cis-thio glycosylations: scope and application to the synthesis of S-linked oligosaccharides, glycolipids, sublancin glycopeptides, and TN/TF antigens.

Studies of S-linked glycoconjugates have attracted growing interest because of their enhanced chemical stability and enzymatic resistance over O-glycoside counterparts. We here report a facile approach to access α-1,2-cis-S-linked glycosides using triflic acid as a catalyst to promote the glycosylation of a series of thiols with d-glucosamine, galactosamine, glucose, and galactose electrophiles. This method is broadly applicable for the stereoselective synthesis of S-linked glycopeptides, oligosaccharides and glycolipids in high yield and excellent α-selectivity. Many of the synthetic limitations associated with the preparation of these S-linked products are overcome by this catalytic method.

Specific Inhibition of Heparanase by a Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.

Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.

Design, synthesis, and evaluation of heparan sulfate mimicking glycopolymers for inhibiting heparanase activity.

Heparanase is an enzyme which cleaves heparan sulfate (HS) polysaccharides of the extracellular matrix. It is a regulator of tumor behavior, plays a key role in kidney related diseases and autoimmune diabetes. We report herein the use of computational studies to extract the natural HS-heparanase interactions as a template for the design of HS mimicking glycopolymers. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor and to have tight-binding characteristics. This glycopolymer also lacks anticoagulant activity.

Glycosidase Inhibition by Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymers.

We report herein the first-time exploration of the attachment of well-defined saccharide units onto a synthetic polymer backbone for the inhibition of a glycosidase. More specifically, glycopolymers endowed with heparan sulfate (HS) disaccharides were established to inhibit the glycosidase, heparanase, with an IC50 value in the low nanomolar range (1.05 ± 0.02 nm), a thousand-fold amplification over its monovalent counterpart. The monomeric moieties of these glycopolymers were designed in silico to manipulate the well-established glycotope of heparanase into an inhitope. Studies concluded that (1) the glycopolymers are hydrolytic stable toward heparanase, (2) longer polymer length provides greater inhibition, and (3) increased local saccharide density (monoantennary vs diantennary) is negligible due to hindered active site of heparanase. Furthermore, HS oligosaccharide and polysaccharide controls illustrate the enhanced potency of a multivalent scaffold. Overall, the results on these studies of the multivalent presentation of saccharides on bottlebrush polymers serve as the platform for the design of potent glycosidase inhibitors and have potential to be applied to other HS-degrading proteins.

Utilization of bench-stable and readily available nickel(II) triflate for access to 1,2-cis-2-aminoglycosides.

The utilization of substoichiometric amounts of commercially available nickel(II) triflate as an activator in the reagent-controlled glycosylation reaction for the stereoselective construction of biologically relevant targets containing 1,2-cis-2-amino glycosidic linkages is reported. This straightforward and accessible methodology is mild, operationally simple and safe through catalytic activation by readily available Ni(OTf)2 in comparison to systems employing our previously in-house prepared Ni(4-F-PhCN)4(OTf)2. We anticipate that the bench-stable and inexpensive Ni(OTf)2, coupled with little to no extra laboratory training to set up the glycosylation reaction and no requirement of specialized equipment, should make this methodology be readily adopted by non-carbohydrate specialists. This report further highlights the efficacy of Ni(OTf)2 to prepare several bioactive motifs, such as blood type A-type V and VI antigens, heparin sulfate disaccharide repeating unit, aminooxy glycosides, and α-GalNAc-Serine conjugate, which cannot be achieved in high yield and α-selectivity utilizing in-house prepared Ni(4-F-PhCN)4(OTf)2 catalyst. The newly-developed protocol eliminates the need for the synthesis of Ni(4-F-PhCN)4(OTf)2 and is scalable and reproducible. Furthermore, computational simulations in combination with 1H NMR studies analyzed the effects of various solvents on the intramolecular hydrogen bonding network of tumor-associated mucin Fmoc-protected GalNAc-threonine amino acid antigen derivative, verifying discrepancies found that were previously unreported.