The deficiency of β-galactosylceramidase causes a lysosomal leukodystrophy, known as Krabbe disease (KD), resulting in elevated psychosine (PSY) levels, which are highly cytotoxic to myelin-forming cells. 2-hydroxypropyl-α-CD (HPaCD), a cyclic-oligosaccharide containing a lipophilic central cavity and hydrophilic outer surfaces, significantly reduces PSY cytotoxicity in cultured KD patient cells. Further 1H-NMR studies revealed stronger interactions between HPaCD and PSY. Regarding safety, HPaCD-treated mice showed no electrophysiological and histological ototoxicity signs. In the murine KD model, HPaCD improved neurobehavior and reduced PSY levels in the CNS and PNS. The reduction of astrogliosis, increased myelin basic protein, and improvements in PNS axonal-myelin morphometrics were also observed in HPaCD-treated mice. In summary, this is an innovative therapeutic approach that leverages HPaCD's dual properties of molecularly shielding and neutralizing PSY and facilitating its CNS and PNS clearance. Since several newborn screening programs currently include KD, HPaCD becomes highly important as an adjunctive/bridge therapy for improving outcomes in this devastating disorder.

Foamy, lipid-laden macrophages are found in multiple sclerosis (MS) lesions, resulting from excessive phagocytosis of myelin debris following demyelination. These lipid-laden macrophages exhibit an inflammatory phenotype, inhibit remyelination, and likely contribute to MS pathology, yet effective therapeutic strategies to target them are lacking. In this study, we sought to better characterize the temporal patterns of myelin debris phagocytosis by human and murine microglia/macrophages, and generate an in vitro model of foamy phagocytes. In addition, given their demonstrated ability to promote lipid efflux in models of other neurological diseases, we investigated cyclodextrins as potential therapeutic agents in MS. We hypothesized that cyclodextrins could lower the accumulation of lipids in foamy macrophages, potentially modulating their inflammatory phenotype. Several in house-synthesized cyclodextrins were evaluated. We found that prolonged application of myelin and inflammatory cytokines produced an inflammatory macrophage phenotype with an MS-like signature, as determined through bulk sequencing analysis. When particular cyclodextrins were applied to these foamy macrophages in culture, there was reduced accumulation of ingested and processed lipids, and altered expression of genes related to inflammatory pathways or wound healing. Our findings suggest that cyclodextrins may modulate the phenotype of inflammatory macrophages, typical of MS pathology, and hold therapeutic potential in MS, warranting further investigation.

The extracellular matrix (ECM) plays an important role in the central nervous system (CNS), shaping tissue structure and functions as well as contributing to the pathology of chronic diseases such as multiple sclerosis (MS). ECM components, including fibulin-2 (FBLN2) and chondroitin sulfate proteoglycans (CSPGs), may impact neuroinflammation and remyelination. We investigated the capacity of FBLN2 to modulate immune responses and evaluated its interaction with CSPGs in experimental autoimmune encephalomyelitis (EAE), a common model for MS. We show that FBLN2 deficiency in EAE mice reduced microglial pro-inflammatory activity, while effects on monocyte-derived macrophages and border-associated macrophages were less pronounced. Targeting FBLN2 and CSPGs individually, using FBLN2-/- mice and the CSPG-synthesis inhibitor difluorosamine (DIF), respectively, enhanced recovery of disability and reduced neuroinflammation in EAE mice. However, their combined targeting did not result in additive therapeutic effects beyond either alone. This study underscores the complex regulatory roles of ECM components on neuroinflammation and provides insights into potential therapeutic strategies for neuroinflammatory diseases.

We report an investigation on the cobalt(II) chelation mechanism by a modified α-maltoside ligand 9 decorated with two iminodiacetate (IDA) residues on C6,C6′ positions. Herein we uncovered the capacity of this biodegradable ligand to chelate cobalt(II), an ionic metal contaminant in the environment that is used, in particular, in lithium-ion batteries. The interactions between cobalt(II) and synthesized ligand 9 were systematically studied using different analytical methods such as 1H and 13C NMR, potentiometry, spectrophotometry, ITC, and ICP-AES. We observed a high affinity for the 1:1 complex, one cobalt(II) associated with two iminodiacetate groups, which is 10-fold higher than the 2:1 complex, where each of the two IDA groups interacts alone with a cobalt(II). Taking into account the log βCoLvalue obtained (≈12.3) with the stoichiometry 1:1, the strength of this complexation with cobalt(II) can be ranked as follows for the most common ligands: IDA < MIDA < NTA < 9 < EDTA < TTHA < DTPA. We further completed a preliminary remediation test with water contaminated with cobalt(II) and recovered cobalt(II) metal using Chelex® resin, which allowed a recycling of the synthetic ligand for future recovering experiments. The results shed light on the great potential of using this synthetic ligand as an effective and green remediation tool.

Intracerebral hemorrhage (ICH) causes prominent deposition of extracellular matrix molecules, particularly the chondroitin sulphate proteoglycan (CSPG) member neurocan. In tissue culture, neurocan impedes the properties of oligodendrocytes. Whether therapeutic reduction of neurocan promotes oligodendrogenesis and functional recovery in ICH is unknown. Mice were retro-orbitally injected with adeno-associated virus (AAV-CRISPR/Cas9) to reduce neurocan deposition after ICH induction by collagenase. Other groups of ICH mice were treated with vehicle or a drug that reduces CSPG synthesis, 4-4-difluoro-N-acetylglucosamine (difluorosamine). Rota-rod and grip strength behavioral tests were conducted over 7 or 14 days. Brain tissues were investigated for expression of neurocan by immunofluorescence microscopy and western blot analysis. Brain cryosections were also stained for microglia/macrophage phenotype, oligodendrocyte lineage cells and neuroblasts by immunofluorescence microscopy. Tissue structural changes were assessed using brain magnetic resonance imaging (MRI). The adeno-associated virus (AAV)-reduction of neurocan increased oligodendrocyte numbers and functional recovery in ICH. The small molecule inhibitor of CSPG synthesis, difluorosamine, lowered neurocan levels in lesions and elevated numbers of oligodendrocyte precursor cells, mature oligodendrocytes, and SOX2+ nestin+ neuroblasts in the perihematomal area. Difluorosamine shifted the degeneration-associated functional state of microglia/macrophages in ICH towards a regulatory phenotype. MRI analyses showed better fiber tract integrity in the penumbra of difluorosamine mice. These beneficial difluorosamine results were achieved with delayed (2 or 3 days) treatment after ICH. Reducing neurocan deposition following ICH injury is a therapeutic approach to promote histological and behavioral recovery from the devastating stroke.

Sucrose constitutes a non-toxic, biodegradable, low-cost and readily available natural product. To expand its utility, we developed total synthesis for a ligand based on a sucrose scaffold for potential use as a metal chelation agent. The designed target (compound 2) has a metal-chelating functionality at both the C-6 and C-6’ positions, which can provide a first coordination sphere of eight valencies. The designed total synthesis was highly efficient. To demonstrate the utility of the ligand, we studied its complexation with Gd(III). Using potentiometric titration and high-resolution mass spectrometry, we confirmed the formation of a 1:1 complex with Gd(III), which has a respectable formation constant of ~1013.4. Further NMR relaxivity studies show that the Gd(III) complex has a relaxivity (r1) of 7.6958 mmol−1 s−1.

In the search for improved and safer gadolinium-based magnetic resonance imaging (MRI) contrast agents, macrocyclic cyclodextrins (CDs) attract great interest. Our group previously synthesized a cyclodextrin-based ligand with 1,2,3-triazolmethyl residues conjugated to β-CD, called β-CD(A), which efficiently chelates Gd(III) ions. To probe the local structure around the Gd(III) ion in the 1:1 Gd(III): β-CD(A) complex in aqueous solution (pH 5.5), we used extended X-ray absorption fine structure (EXAFS) spectroscopy. Least-squares curve fitting of the Gd L3-edge EXAFS spectrum revealed 5 Gd–O (4 COO− and 1 H2O) and 4 Gd–N (from two imino and two 1,2,3-triazole groups) bonds around the Gd(III) ion with average distances 2.36 and 2.56 ± 0.02 Å, respectively. A similar EXAFS spectrum was obtained from an aqueous solution of the clinically used MRI contrast agent Na[Gd(DOTA)(H2O)], also 9-coordinated in its first shell. Careful analysis revealed that the mean Gd–N distance is shorter in the Gd(III): β-CD(A) (1:1) complex, indicating stronger Gd–N bonding and stronger Gd(III) complex formation than with the DOTA4− ligand. This is consistent with the lower free Gd3+ concentration found previously for the Gd(III): β-CD(A) (1:1) complex than for the [Gd(DOTA)(H2O)]− complex, and shows its potential as an MRI probe. EXAFS spectroscopy revealed a similar Gd(III) 9-coordination although slightly stronger for a modified β-cyclodextrin: Gd(III) 1:1 complex, [Gd(LH4)]7−, in aqueous solution than for the clinically used MRI contrast agent Na[Gd(DOTA)(H2O)].

Campylobacter jejuni is a bacterial pathogen that causes hundreds of millions of cases of food-borne gastroenteritis worldwide annually. The infection caused by this bacterium is also associated with several forms of post-infectious autoimmune sequelae that can be very serious, including the life-threatening Guillain-Barré syndrome. The capsular polysaccharides (CPS) of C. jejuni HS:4 consist of a very unique repeating disaccharide unit that is characterized by a β-1,4-linked 6-deoxy-β-D-ido-heptopyranose and an N-acetyl-β-D-glucosamine. Eliciting carbohydrate-specific antibodies against the CPS structures of C. jejuni HS:4 is an attractive strategy. The 6-deoxy-ido-configuration of the heptose combined with its β-anomeric configuration makes the chemical synthesis of the disaccharide very challenging. Here, we report an efficient synthesis to obtain the key repeating disaccharide and its analog in reverse order plus a trisaccharide. Our synthesis features a highly efficient, one-step stereo- and regioselective conversion of β-D-galacto-heptopyranosides to 6-deoxy-β-D-ido-heptopyranosides via the intermediate 2,3-anhydro-β-D-talo-heptopyranosides.

Systemic treatment of cancer using long-circulating nanomedicines is promising due to their passive tumor targeting ability to achieve higher and more selective accumulation in tumors with irregular vascularization, a phenomenon known as extended permeation and retention (EPR) effect.1,2 Clinical use of nanometer-sized carriers, such as Doxil and Abraxane, to deliver chemotherapeutics to solid tumors is proven effective in highly vascularized tumors such as breast cancer, ovarian cancer, multiple myeloma, and Kaposi9s sarcoma.3-5 Most nanomedicines that are being developed or approved so far have a diameter of around 100-200 nm for prolonged retention in highly angiogenic and densely vascularized tumors,6 however, they suffer from limited accumulation and poor penetration to the inner core of avascular or hypovascular tumors (such as prostate and pancreatic cancer),7-9 therefore nanomedicines small than 100 nm are more preferred for improved tumor penetration.10,11 Here we present our strategy to form cyclodextrin-based sub-30-nm nanocarriers, which allows easy drug encapsulation, and successful delivery of therapeutics to human tumor xenografts with significantly reduced tumor growth rates and improved survival rates. Citation Format: Xiaowei Ma, Ping Zhang, Chao Cui, Chang-Chun Ling, Lina Cui. Sub-30-nm capsules for drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 280.

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate-based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. The display of glycans on each phage virion can be controlled from 30-1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec-2) and DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), and in live mice (Nat. Chem. Bio. 17, 806-816, 2021). From a mixture of 50-100 multivalent glycan probes, LiGA identifies the glycan-phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo. Sialic acid-binding immunoglobulin-type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec-7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec-1 using a cell-based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans- glycan to overcome the cis- masking on the surface of immune cells.

Bacterial nonulosonic acids such as pseudaminic acids and others constitute a family of 9-carbon monosaccharides that contain a common 3-deoxy-2-ketoacid fragment but differ in their stereochemistries at 5 stereogenic centers between C-4 to C-8. Their unique structures make them attractive targets for use as antigens in vaccinations to combat drug-resistant bacterial infections and their challenging stereochemistries have attracted considerable attention from chemists. In this work we report the development of an improved synthesis for 2,4-di-N-acetyl-l-altrose (l-2,4-Alt-diNAc), which is a key hexose required for the chemical and chemoenzymatic synthesis of pseudaminic acids. Using l-fucose as a starting material, our synthesis overcomes several pitfalls in previously reported syntheses.

The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a “Liquid Glycan Array” (LiGA) platform comprising a library of DNA ‘barcoded’ M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo ; measurements that cannot be performed with canonical glass slide-based glycan arrays. Dedication The paper is dedicated to Laura L. Kiessling on the occasion of her 60th birthday.

Here, we report the synthesis and detailed studies on the coordination chemistry of a novel chemically modified polyaminocarboxylate (5) based on β-cyclodextrin (CD) scaffold for lanthanides. The target ligand is prepared in a highly efficient manner (seven total steps) from β-CD using the readily available iminodiacetic acid as a starting material. A propargyl group is attached to the iminodiacetate via N-alkylation, and the obtained derivative is efficiently conjugated to the β-CD scaffold via the copper(I)-mediated 1,3-dipolar cycloaddition. The generated 1,2,3-triazolmethyl residues advantageously provide a competent chelating group while displacing the metal coordination center away from the primary rim of β-CD, to afford the required conformational flexibility. The functional groups from each of the two adjacent glucopyranosyl units of β-CD complete a uniquely created octavalent coordination sphere for lanthanides while still sparing one site for dynamic water coordination. To help study the coordination chemistry of CD ligand 5, we also design a relevant maltoside ligand 6, which faithfully represents one submetal-binding section of ligand 5. Thanks to HRMS and NMR studies, we successfully elucidate the coordination chemistries of synthesized ligands. The octavalent coordination sphere of ligand 5 shows strong binding affinity to lanthanides. By potentiometric titration experiments, ligand 5 is found to bind gadolinium(III), forming 1:1, 1:2, and 1:3 multinuclear complexes with lanthanides, thus possessing great capacity for catalyzing the dynamic water-exchange. Further NMR studies also reveal that the formed ligand 5/Gd(III) complexes show significantly better abilities to alter T1 relaxivities of coordinated water than DOTA-Gd(III) and also some of the synthetic CD probes reported in the literature.

Using amphiphilic cyclodextrin as a scaffold, the first class of PK-glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.

The synthesis of novel cyanoethylated cyclodextrin derivatives from previously prepared intermediates is described. It was found the alcohols at the primary face of cyclodextrins readily react to add to acrylonitrile, but similar additions from hydroxyl groups of the secondary face appear to be more difficult. The obtained cyanides could be reduced to form the corresponding amines.

A β-GlcNAc-LeX tetrasaccharides and another α-Gal-LeX analog have been synthesized for studying interaction with toxins produced by the human pathogen Clostridium difficile. LeX-based trisaccharides bearing either a 2-azidoethyl or a 6-azidohexyl aglycone have been employed in the total synthesis to afford the desired tetrasaccharides. Interestingly, during the final catalytic hydrogenation step to remove benzyl and benzylidene protecting groups and simultaneously reduce azide functionality, partial N-methylations were observed. The N-methylations appear to be a general issue with catalytic hydrogenation of azides in methanol.

The ability to form self-organized thermotropic mesophases of amphiphilic cyclodextrins correlates well with their ability to establish an intermolecular H-bond network.

OBJECTIVE: To assess the preclinical efficacy of a fluorinated analog of acetylated N-acetylglucosamine as a therapy to promote myelin repair in multiple sclerosis by normalizing the expression of inhibitory chondroitin sulfate proteoglycan barrier molecules. BACKGROUND: Remyelination, the generation of new myelin after CNS insult, is impeded largely by the inhibitory nature of the extracellular lesion microenvironment. Our group recently established that the chondroitin sulfate proteoglycans (CSPGs), which are upregulated in CNS conditions such as multiple sclerosis, are potent inhibitors of myelin repair by oligodendrocyte precursor cells (OPCs) (Lau et al., Ann Neurol 72:419-432, 2012). We aimed to reduce this aberrant CSPG with a fluorinated analog of acetylated N-acetylglucosamine (Ac-4-F-GlcNAc), a synthesis inhibitor. DESIGN/METHODS: Murine astrocytes, which produce CSPGs when stimulated, were cultured in the presence of Ac-4-F-GlcNAc. Treated media was analyzed for CSPG content as well as functional effects on OPCs in culture. Ac-4-F-GlcNAc was administered following dorsal column microinjections of the demyelinating detergent lysolecithin in mice, and the content of spinal cord CSPGs was later assessed. Ac-4-F-GlcNAc was also administered to mice afflicted with experimental autoimmune encephalomyelitis (EAE) to assess the effects on an inflammatory model of MS. RESULTS: Murine OPCs are potently inhibited in terms of cell adhesion and process outgrowth in the presence of CSPGs. Ac-4-F-GlcNAc dramatically reduces the CSPG content of astrocytes in culture without evidence of toxicity, and OPCs appear less inhibited in the presence of this media. In vivo, Ac-4-F-GlcNAc reduces the CSPG content of mouse spinal cords following lysolecithin, and reduces the severity of EAE. CONCLUSIONS: Ac-4-F-GlcNAc appears to be a safe, systemic approach to reduce CSPGs following demyelination as well as mitigate the inflammatory component of EAE. We are currently addressing whether Ac-4-F-GlcNAc results in improved OPC recruitment and maturation in the lesion environment, and ultimately the impact on remyelination. Study Supported by: Multiple Sclerosis Society of Canada

A highly valuable tri-/tetra-functionalization method is reported for cyclodextrin (CD) chemistry by taking advantage of the acid-sensitivity of the O-silyl ether group and also the geometry of CD macrocycles. The controlled acid-mediated. O-desilylations from the easily accessible per-3,6-O-silylaled CD derivatives provide unprecedented regioselectivity to differentiate not only primary O-silyl groups from secondary groups, but also O-silyl groups of the same type with identical chemical reactivity. This methodology differs from other conventional monofunctionalization methods of natural CDs, which only allow for a direct twofold differentiation of hydroxyl groups in a CD, because the current method permits the synthesis of and tetra-functionalized CDs in a short reaction sequence. Most importantly, the developed method has been found to be applicable to all alpha-, beta-, and gamma-CDs and the obtained CD intermediates are versatile. Furthermore, we demonstrate that these processes are practical and can be carried out on multigram scales.