Sulfated disaccharide protects membrane and DNA damages from arginine-rich dipeptide repeats in ALS.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. Here, we examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. We screened and identified a sulfated disaccharide that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, our results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS.

An integrated microfluidic system for automatic detection of cholangiocarcinoma cells from bile.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

The collision-induced dissociation mechanism of sodiated Hex-HexNAc disaccharides.

Determining carbohydrate structures, such as their compositions, linkage positions, and in particular the anomers and stereoisomers, is a great challenge. Isomers of different anomers or stereoisomers have the same sequences of chemical bonds, but have different orientations of some chemical bonds which are difficult to be distinguished by mass spectrometry. Collision-induced dissociation (CID) tandem mass spectroscopy (MS/MS) is a widely used technique for characterizing carbohydrate structures. Understanding the carbohydrate dissociation mechanism is important for obtaining the structural information from MS/MS. In this work, we studied the CID mechanism of galactose-N-acetylgalactosamine (Gal-GalNAc) and glucose-N-acetylglucosamine (Glc-GlcNAc) disaccharides with 1→3 and 1→4 linkages. For Gal-GalNAc disaccharides, the CID mass spectra of sodium ion adducts show significant difference between the α- and ß-anomers of GalNAc at the reducing end, while no difference in the CID mass spectra between two anomers of Glc-GlcNAc disaccharides was found. Quantum chemistry calculations show that for Gal-GalNAc disaccharides, the difference of the dissociation barriers between dehydration and glycosidic bond cleavage is significantly small in the ß-anomer compared to that in the α-anomer; while these differences are similar between the α- and ß-anomers of Glc-GlcNAc disaccharides. These differences can be attributed to the different orientations of hydroxyl and N-acetyl groups located at GalNAc and GlcNAc. The calculation results are consistent with the CID spectra of isotope labelled disaccharides. Our study provides an insight into the CID of 1→3 and 1→4 linked Gal-GalNAc and Glc-GlcNAc disaccharides. This information is useful for determining the anomeric configurations of GalNAc in oligosaccharides.

The Good, the Bad, and the Ugly Memories of Carbohydrate Fragments in Collision-Induced Dissociation.

Collision-induced dissociation (CID) tandem mass spectrometry is commonly used for carbohydrate structural determinations. In the CID tandem mass spectrometry approach, carbohydrates are dissociated into fragments, and this is followed by the structural identification of fragments through subsequent CID. The success of the structural analysis depends on the structural correlation of fragments before and after dissociation, that is, structural memory of fragments. Fragments that completely lose the memory of their original structures cannot be used for structural analysis. By contrast, fragments with extremely strong correlations between the structures before and after fragmentation retain the information on their original structures as well as have memories of their precursors' entire structures. The CID spectra of these fragments depend on their own structures and on the remaining parts of the precursor structures, making structural analysis impractical. For effective structural analysis, the fragments produced from a precursor must have good structural memory, meaning that the structures of these fragments retain their original structure, and they must not be strongly affected by the remaining parts of the precursors. In this study, we found that most of the carbohydrate fragments produced by low-energy CID have good memory in terms of linkage position and anomericity. Fragments with ugly memory, where fragment structures change with the remaining parts of the precursors, can be attributed to C ion formation in a linear form. Fragments with ugly memory can be changed to have good memory by preventing linear C ion generation by using an alternative CID sequence, or the fragments of ugly memory can become useful in structural analysis when the contribution of linear C ions in fragmentation patterns is understood.

A concise synthesis of l-gulose and its C-6 derivatives.

A convenient route for the preparation of l-gulose and its C-6 derivatives starting from commercially available 2,3:5,6-diisopropylidene-d-mannofuranose via C-5 epimerization as the key step was developed. 1-O-Benzylation followed by regioselective hydrolysis of the 5,6-isopropylidene group furnished benzyl 2,3-isopropylidene-α-d-mannofuranoside, which was subjected upon regioselective one-pot 6-O-benzoylation and 5-O-mesylation, providing the corresponding 5-OMs-6-OBz derivative in excellent selectivity. Treatment of this mesylate compound with potassium t-butoxide to remove the benzoyl group followed by intramolecular SN2 inversion led to benzyl 5,6-anhydro-2,3-isopropylidene-ß-l-gulofuranoside, which could undergo not only nucleophilic substitutions to open the epoxide ring to give various C-6 derivatives, but also acidic hydrolysis to yield 1,6-anhydro-ß-l-gulopyranose for further transformation into l-gulopyranosyl pentaacetate.

Design and Synthesis of 1-O- and 6'-C-Modified Heparan Sulfate Trisaccharides as Human Endo-6-O-Sulfatase 1 Inhibitors.

The extracellular human endo-6-O-sulfatases (Sulf-1 and Sulf-2) are responsible for the endolytic cleavage of the 6-sulfate groups from the internal D-glucosamine residues in the highly sulfated subdomains of heparan sulfate proteoglycans. A trisaccharide sulfate, IdoA2OS-GlcNS6S-IdoA2OS, was identified as the minimal size of substrate for Sulf-1. In order to study the complex structure with Sulf-1 for developing potential drugs, two trisaccharide analogs, IdoA2OS-GlcNS6OSO2NH2-IdoA2OS-OMe and IdoA2OS-GlcNS6NS-IdoA2OS-OMe, were rationally designed and synthesized as the Sulf-1 inhibitors with IC50 values at 0.27 and 4.6 µM, respectively.

Byproduct formation during the biosynthesis of spinosyn A and evidence for an enzymatic interplay to prevent its formation.

Biosynthesis of spinosyn A in Saccharopolyspora spinosa involves a 1,4-dehydration followed by an intramolecular [4 + 2]-cycloaddition catalyzed by SpnM and SpnF, respectively. The cycloaddition also takes place in the absence of SpnF leading to questions regarding its mechanism of catalysis and biosynthetic role. Substrate analogs were prepared with an unactivated dienophile or an acyclic structure and found to be unreactive consistent with the importance of these features for cyclization. The SpnM-catalyzed dehydration reaction was also found to yield a byproduct corresponding to the C11 = C12 cis isomer of the SpnF substrate. This byproduct is stable both in the presence and absence of SpnF; however, relative production of the SpnM product and byproduct could be shifted in favor of the former by including SpnF or the dehydrogenase SpnJ in the reaction. This result suggests a potential interplay between the enzymes of spinosyn A biosynthesis that may help to improve the efficiency of the pathway.

Unusual free oligosaccharides in human bovine and caprine milk.

Free oligosaccharides are abundant macronutrients in milk and involved in prebiotic functions and antiadhesive binding of viruses and pathogenic bacteria to colonocytes. Despite the importance of these oligosaccharides, structural determination of oligosaccharides is challenging, and milk oligosaccharide biosynthetic pathways remain unclear. Oligosaccharide structures are conventionally determined using a combination of chemical reactions, exoglycosidase digestion, nuclear magnetic resonance spectroscopy, and mass spectrometry. Most reported free oligosaccharides are highly abundant and have lactose at the reducing end, and current oligosaccharide biosynthetic pathways in human milk are proposed based on these oligosaccharides. In this study, a new mass spectrometry technique, which can identify linkages, anomericities, and stereoisomers, was applied to determine the structures of free oligosaccharides in human, bovine, and caprine milk. Oligosaccharides that do not follow the current biosynthetic pathways and are not synthesized by any discovered enzymes were found, indicating the existence of undiscovered biosynthetic pathways and enzymes.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Practical Remdesivir Synthesis through One-Pot Organocatalyzed Asymmetric (S)-P-Phosphoramidation.

Remdesivir, an inhibitor of RNA-dependent RNA polymerase developed by Gilead Sciences, has been used for the treatment of COVID-19. The synthesis of remdesivir is, however, challenging, and the overall cost is relatively high. Particularly, the stereoselective assembly of the P-chirogenic center requires recrystallization of a 1:1 isomeric p-nitrophenylphosphoramidate mixture several times to obtain the desired diastereoisomer (39%) for further coupling with the d-ribose-derived 5-alcohol. To address this problem, a variety of chiral bicyclic imidazoles were synthesized as organocatalysts for stereoselective (S)-P-phosphoramidation employing a 1:1 diastereomeric mixture of phosphoramidoyl chloridates as the coupling reagent to avoid a waste of the other diastereomer. Through a systematic study of different catalysts at different temperatures and concentrations, a mixture of the (S)- and (R)-P-phosphoramidates was obtained in 97% yield with a 96.1/3.9 ratio when 20 mol % of the chiral imidazole-cinnamaldehyde-derived carbamate was utilized in the reaction at -20 °C. A 10-g scale one-pot synthesis via a combination of (S)-P-phosphoramidation and protecting group removal followed by one-step recrystallization gave remdesivir in 70% yield and 99.3/0.7 d.r. The organocatalyst was recovered in 83% yield for reuse, and similar results were obtained. This one-pot process offers an excellent opportunity for industrial production of remdesivir.

Identification of existing pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection.

The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 µM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti-SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.

Concise and Reliable Syntheses of Glycodendrimers via Self-Activating Click Chemistry: A Robust Strategy for Mimicking Multivalent Glycan-Pathogen Interactions.

Individual interactions between glycans and their receptors are usually weak, although these weak interactions can combine to realize a strong interaction (multivalency). Such multivalency plays a crucial role in the recognition of host cells by pathogens. Glycodendrimers are useful materials for the reconstruction of this multivalent interaction. However, the introduction of a large number of glycans to a dendrimer core is fraught with difficulties. We herein synthesized antipathogenic glycodendrimers using the self-activating click chemistry (SACC) method developed by our group. The excellent reactivity of SACC enabled the efficient preparation of sialyl glycan and Gb3 glycan dendrimers, which exhibited strong avidity toward hemagglutinin on influenza virus and Shiga toxin B subunit produced by Escherichia coli, respectively. We demonstrated the usefulness of SACC-based glycodendrimers as antipathogenic compounds.

Synthesis of hyaluronic acid oligosaccharides with a GlcNAc-GlcA repeating pattern and their binding affinity with CD44.

Hyaluronic acid (HA) is a ubiquitous glycosaminoglycan in the extracellular matrix and a ligand of CD44, a transmembrane glycoprotein that is important in cell migration. Crystal and NMR studies found a hexasaccharide of the pattern (GlcA-GlcNAc)3 as the shortest HA that could bind to CD44, but molecular dynamics simulations indicated that a tetrasaccharide of the pattern (GlcNAc-GlcA)2 is the key structure interacting with CD44. Access to oligomers with such a repeat pattern is crucial in binding studies with CD44. Here we developed a synthetic procedure to afford the HA oligosaccharides with the GlcNAc-GlcA repeating unit and measured the binding interaction between these sugars and human CD44 by isothermal titration calorimetry (ITC). During the chemical synthesis, we successfully generated the ß-glycosidic bond in the absence of neighbouring group participation and overcome the issues in the oxidation step. In addition, ammonia-free dissolving metal reduction for debenzylation and azido reduction has been applied in carbohydrate synthesis for the first time. ITC analysis revealed that the HA tetrasaccharide (GlcNAc-GlcA)2 could indeed interact and bind to the human CD44.

Exploring Circulating Tumor Cells in Cholangiocarcinoma Using a Novel Glycosaminoglycan Probe on a Microfluidic Platform.

The search of alternative approaches to epithelial cell adhesion molecule (EpCAM), for the isolation of circulating tumor cells (CTC), is on the rise. This work attempts at evaluating the feasibility of using a new glycosaminoglycan, SCH45, as a probe to isolate CTCs from the peripheral blood of 65 advanced/metastatic cholangiocarcinoma (CCA) patients. The positive enrichment of CTCs from 1 mL of blood using SCH45-bound magnetic beads and subsequent staining on an integrated microfluidic platform is demonstrated. Results detailing CTC concentrations averaging ≥1 CTCs mL-1 of blood are shown, and a conventional protein biomarker, EpCAM, has been used to corroborate the finding that 100% of the patients possess CTCs in their blood. Studies detailing the use of CTCs in the prognostic monitoring and treatment effectiveness of advanced/metastatic CCA are scarce, and the isolation of CTCs from all CCA patients tested has not been reported yet. A strong correlation between CTC counts and disease progression at the time of and/or in advance of radiographic imaging in patients receiving chemotherapy is also reported. This study is one of its kind with the new probe and reduced sample volume and has potential for use in CCA diagnosis and prognosis in the near future.

Trisaccharide Sulfate and Its Sulfonamide as an Effective Substrate and Inhibitor of Human Endo-O-sulfatase-1.

Human endo-O-sulfatases (Sulf-1 and Sulf-2) are extracellular heparan sulfate proteoglycan (HSPG)-specific 6-O-endosulfatases, which regulate a multitude of cell-signaling events through heparan sulfate (HS)-protein interactions and are associated with the onset of osteoarthritis. These endo-O-sulfatases are transported onto the cell surface to liberate the 6-sulfate groups from the internal d-glucosamine residues in the highly sulfated subdomains of HSPGs. In this study, a variety of HS oligosaccharides with different chain lengths and N- and O-sulfation patterns via chemical synthesis were systematically studied about the substrate specificity of human Sulf-1 employing the fluorogenic substrate 4-methylumbelliferyl sulfate (4-MUS) in a competition assay. The trisaccharide sulfate IdoA2S-GlcNS6S-IdoA2S was found to be the minimal-size substrate for Sulf-1, and substitution of the sulfate group at the 6-O position of the d-glucosamine unit with the sulfonamide motif effectively inhibited the Sulf-1 activity with IC50 = 0.53 µM, Ki = 0.36 µM, and KD = 12 nM.

Sulfation pattern of chondroitin sulfate in human osteoarthritis cartilages reveals a lower level of chondroitin-4-sulfate.

Chondroitin sulfates (CS) account for more than 80% of the glycosaminoglycans of articular cartilage, which impart its physiological functions. We quantified the absolute concentration of the CS components of the full thickness cartilages from the knees of patients with terminal-phase osteoarthritis. Osteochondrol biopsies were removed from the medial femoral condyle and lateral femoral condyle of sixty female patients received total knee arthroplasty, aged from 58 to 83 years old. We found the total CS concentrations and chondroitin-4-sulfate disaccharide were significantly lowered in osteoarthritic samples. Microstructure analysis indicated while chondroitin-0-sulfate was equally distributed across different zones of the osteoarthritic cartilages, chondroitin-4-sulfate is significantly less in the deep zones. Down-regulation of sulfotransferases, the enzymes responsible for CS sulfation, in the lesion site of cartilage were observed. Our study suggested chondroitin-4-sulfate down-regulation can be a diagnostic marker for degraded osteoarthritis cartilage, with potential implications in cartilage regeneration.

Glycan sulfation patterns define autophagy flux at axon tip via PTPRσ-cortactin axis.

Chondroitin sulfate (CS) and heparan sulfate (HS) are glycosaminoglycans that both bind the receptor-type protein tyrosine phosphatase PTPRσ, affecting axonal regeneration. CS inhibits axonal growth, while HS promotes it. Here, we have prepared a library of HS octasaccharides and, together with synthetic CS oligomers, we found that PTPRσ preferentially interacts with CS-E-a rare sulfation pattern in natural CS-and most HS oligomers bearing sulfate and sulfamate groups. Consequently, short and long stretches of natural CS and HS, respectively, bind to PTPRσ. CS activates PTPRσ, which dephosphorylates cortactin-herein identified as a new PTPRσ substrate-and disrupts autophagy flux at the autophagosome-lysosome fusion step. Such disruption is required and sufficient for dystrophic endball formation and inhibition of axonal regeneration. Therefore, sulfation patterns determine the length of the glycosaminoglycan segment that bind to PTPRσ and define the fate of axonal regeneration through a mechanism involving PTPRσ, cortactin and autophagy.

An integrated microfluidic system for rapid detection and multiple subtyping of influenza A viruses by using glycan-coated magnetic beads and RT-PCR.

The influenza A (InfA) virus, which poses a significant global public health threat, is routinely classified into "subtypes" based on viral hemagglutinin (HA) and neuraminidase (NA) antigens. Because there are nearly 200 viral subtypes, current diagnostic approaches require multiplexing or array systems to cover various subtypes of HA and NA. A microfluidic chip featuring a HA × NA array was consequently developed herein for diagnosis and subtyping of InfA viruses via the use of glycan-coated magnetic beads followed by reverse transcription (RT) polymerase chain reaction (PCR). Up to 12 InfA subtypes were simultaneously detected in an automated fashion in less than 100 minutes on this microfluidic platform, representing a significant improvement in analysis speed compared to benchtop RT-PCR and chip-based microarray systems. The limits of detection of the RT-PCR assays ranged from 40 to 3000 copy numbers for the different subtypes of InfA viruses, around two orders of magnitude higher than in previous studies using microfluidic technologies. In summary, the array-type microfluidic chip system provides a rapid, sensitive, and fully automated approach for detection and multiple subtyping of InfA.

Development of α-Gal-Antibody Conjugates to Increase Immune Response by Recruiting Natural Antibodies.

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.

Selective Inhibition of PAR4 (Protease-Activated Receptor 4)-Mediated Platelet Activation by a Synthetic Nonanticoagulant Heparin Analog.

Objective- PAR4 (protease-activated receptor 4), one of the thrombin receptors in human platelets, has emerged as a promising target for the treatment of arterial thrombotic disease. Previous studies implied that thrombin exosite II, known as a binding site for heparin, may be involved in thrombin-induced PAR4 activation. In the present study, a heparin octasaccharide analog containing the thrombin exosite II-binding domain of heparin was chemically synthesized and investigated for anti-PAR4 effect. Approach and Results- PAR4-mediated platelet aggregation was examined using either thrombin in the presence of a PAR1 antagonist or γ-thrombin, which selectively activates PAR4. SCH-28 specifically inhibits PAR4-mediated platelet aggregation, as well as the signaling events downstream of PAR4 in response to thrombin. Moreover, SCH-28 prevents thrombin-induced ß-arrestin recruitment to PAR4 but not PAR1 in Chinese Hamster Ovary-K1 cells using a commercial enzymatic complementation assay. Compared with heparin, SCH-28 is more potent in inhibiting PAR4-mediated platelet aggregation but has no significant anticoagulant activity. In an in vitro thrombosis model, SCH-28 reduces thrombus formation under whole blood arterial flow conditions. Conclusions- SCH-28, a synthetic small-molecular and nonanticoagulant heparin analog, inhibits thrombin-induced PAR4 activation by interfering with thrombin exosite II, a mechanism of action distinct from other PAR4 inhibitors that target the receptor. The characteristics of SCH-28 provide a new strategy for targeting PAR4 with the potential for the treatment of arterial thrombosis.